Bio-synthesis and characterization of silver nanoparticles from Trichoderma species against cassava root rot disease

Cassava root rot disease caused by the fungal pathogens Fusarium solani and Lasiodiplodia theobromae produces severe damages on cassava production. This research was conducted to produce and assess silver nanoparticles (AgNPs) synthesized by Trichoderma harzianum for reducing root rot disease. The results revealed that using the supernatants of T. harzianum on a silver nitrate solution changed it to reddish color at 48 h, indicating the formation of AgNPs. Further characterization was identified using dynamic light scattering (DLS) and scanning electron microscope (SEM). DLS supported that the Z-average size is at 39.79 nm and the mean zeta potential is at − 36.5 mV. SEM revealed the formation of monodispersed spherical shape with a diameter between 60–75 nm. The antibacterial action of AgNPs as an antifungal agent was demonstrated by an observed decrease in the size of the fungal colonies using an increasing concentration of AgNPs until the complete inhibition growth of L. theobromae and F. solani at > 58 µg mL−1 and at ≥ 50 µg mL−1, respectively. At in vitro conditions, the applied AgNPs caused a decrease in the percentage of healthy aerial hyphae of L. theobromae (32.5%) and of F. solani (70.0%) compared to control (100%). The SR-FTIR spectra showed the highest peaks in the first region (3000–2800 cm−1) associated with lipids and fatty acids located at 2962, 2927, and 2854 cm−1 in the AgNPs treated samples. The second region (1700–1450 cm−1) consisting of proteins and peptides revealed the highest peaks at 1658, 1641, and 1548 cm−1 in the AgNPs treated samples. The third region (1300–900 cm−1), which involves nucleic acid, phospholipids, polysaccharides, and carbohydrates, revealed the highest peaks at 1155, 1079, and 1027 cm−1 in the readings from the untreated samples. Finally, the observed root rot severity on cassava roots treated with AgNPs (1.75 ± 0.50) was significantly lower than the control samples (5.00 ± 0.00).


Biogenic synthesis of silver nanoparticles
The preparation of AgNPs was conducted in two different conditions.For the first one, 5 g of biomass was transferred to 100 mL of 5 mM silver nitrate (AgNO 3 , analytical reagent grade, QReC, New Zealand) solution.For the second, 100 mL of extracellular metabolites culture was mixed with 100 mL of 5 mM AgNO 3 , then incubated in darkness until it experienced color change from light yellow to dark brown, which is an indication of the formation of AgNPs 7 .The AgNPs was separated using centrifugation at 8500 rpm for 15 min and rinsed with deionized water two times.Finally, the reactive mixture containing AgNPs was freeze-dried to completely remove the water contents 25 .

Dynamic light scattering
The size and zeta potential of the AgNPs were measured by using a Zetasizer Nano ZS analyzer (Malvern Instruments Ltd., Worcestershire, UK).The AgNPs were diluted in water at 25 °C then loaded into the Zetasizer Nano ZS.The light from the laser emitter illuminated the sample in order to measure the dynamic fluctuations of light scattering intensity caused by the motion of the particles 26 .

Scanning electron microscope (SEM)
The surface morphology of the AgNPs was observed by using Scanning electron microscope (SEM) (JEOL JSM-6010LV, Japan).The AgNPs suspension was deposited on pin stubs 27 .The images of the size and morphology of AgNPs were recorded.

Field emission transmission electron microscopy (FE-TEM)/scanning transmission electron microscopy-energy dispersive X-ray spectroscopy (STEM-EDS) analysis
To determine the size, shape, and elemental composition of synthesized nanoparticles, 10 µL of the sample was applied onto on copper grids coated with formvar/carbon and allowed to dry overnight, then subjected to FE-TEM/STEM-EDS (Thermos Scientific TALOS F200X, USA) at 200 kV.The FE-TEM was used to capture highresolution images of the AgNPs and STEM-EDS was used to obtain compositional information.

Determination of inhibition zone by agar plate assay
Four different concentrations of bio-synthetized AgNPs at 20, 30, 40, and 50 µg mL −1 were used along with two positive controls (AgNO 3 at 100 µg mL −1 , and carbendazim at 4 mg mL −1 ) and one negative control (water).The antimicrobial activity was determined by the agar plate assay 28 .100 µL of each previously described solutions were spread on a PDA agar plate; then, an 8-mm-agar disc of the mycelium of pathogen was deposited on each plate center and incubated at 25 ± 2 °C.The fungal mycelium was measured at 7 days post incubation.The results were recorded as a percentage value correlated to mycelium growth inhibition using the following simple equation: where A and B were the maximum and the minimum values of the mycelium, respectively.

Minimum inhibitory concentration (MIC) assay
The MIC of the AgNPs was determined in aseptic 96-well plates.One hundred microliters of spore suspension containing each F. solani and L. theobromae with a density of 1 × 10 5 spores mL −1 in PDB was added to 100 μL of PDB containing the AgNPs in different final concentrations from 40 to 60 µg mL −1 .The spore suspension was stored at 25 ± 2 °C for 72 h in the E24 incubator (New Brunswick, Germany).The inhibition of growth was determined by measuring the light absorbance of the mixture at 750 nm by using the Epoch micro-plate spectrophotometer (Biotek, Germany) 29,30 .

In vitro culture of Lasiodiplodia theobromae and Fusarium solani with AgNPs
In order to test the toxicity of AgNPs, the samples were introduced into the PDA in three different concentrations (0, 40, and 50 µg mL −1 ).Two milliliters of L. theobromae and F. solani spore suspension (1 × 10 5 spores mL −1 ) were amended onto the medium and incubated at 28 ± 2 °C for 12 h under fluorescent light and 12 h under darkness.Subsequently, the aerial hyphae growth was observed under light microscopy.

Biochemical changes of fungi mycelium by Synchrotron radiation-based Fourier-transform infrared (SR-FTIR) microspectroscopy
The biochemical changes of the pathogens mycelium L. theobromae and F. solani treated with bio-synthetized AgNPs as well as the positive and negative control samples were analyzed by using SR-FTIR microspectroscopy.The F. solani growth on PDA was incubated at 28 ± 2 °C for ten days and the L. theobromae on PDA was incubated at 28 ± 2 °C for seven days.Later, a 8 mm diameter cork borer was used to drill at the edges of the pathogen colonies and transfer the samples to the PDB amended with two concentrations of biosynthesized AgNPs (40 and 50 µg mL −1 ), AgNO 3 as a positive control (50 µg mL −1 ), and untreated.Next, the pathogen mycelium was incubated at 28 ± 2 °C for 72 h.The mycelium were collected by centrifugation (Thermo Scientific, Germany) at 8500×g, 4 °C for 15 min, and then washed twice with nuclease free water.A mass of mycelium was placed into barium fluoride windows (BaF 2 ) 31 .The mycelium were dehydrated in vacuum for 24 h; then, subjected to SR-FTIR microspectroscopy.The spectral data of each sample was collected with a resolution of 6 cm −1 and a scan time of 64 at the beamline 4.1 Infrared Spectroscopy and Imaging, the Synchrotron Light Research Institute (SLRI), Thailand.The obtained spectral data were analyzed using the OPUS 7.5 software (Bruker Ltd., Germany).Using the Unscrambler X 10.5 software, the original spectra were converted to second derivatives and vector normalized unity for multivariate statistical analysis.This experiment was performed in four replications and repeated three times.

Effect of bio-synthesis AgNPs to control cassava root rot disease
In order to determine the potential of biosynthesized AgNPs on limiting L. theobromae and F. solani, the adopted evaluation procedure was slightly adapted from Onyeka et al. 32 and Boas et al. 33 .Whole cassava root cv.CMR 89 from the Department of Agriculture, Ministry of Agriculture and Cooperatives, NakhonRatchasima, Thailand was sterilized with 1% of sodium hypochlorite (NaClO) solution and washed twice with distilled water.Then, an 8 mm of cork borer was used to make uniform holes of 4 mm in depth of the root.The root samples of each treatment were sprayed with 10 mL of bio-synthetized AgNPs, positive control, and negative control, then left until dry in a laminar flow.Afterwards, 100 µL of fungal spore suspension with density of 1 × 10 5 spores mL −1 was dropped on the drilling points on the roots 32,33 .The roots were kept in a container containing moistened cotton; then, incubated for 10 days.The disease symptoms appearing on the tested root samples were analyzed and rated based on a scale of disease severity for the cassava root rot disease, specified as follows: 1 = no mycelia formation, 2 = 1-25% mycelia formation, 3 = 26-50% mycelia formation, 4 = 51-75% mycelia formation, and 5 = mycelia covering the surface of the slice.

Statistical analysis
All experiments were repeated four times, and the average data for each experiment and replication were reported.After being analyzed the data, an analysis of variance (ANOVA) was performed on it (SPSS software, version 19).The magnitude of the F value (P = 0.05) was used to assess the significance of the different treatments.Duncan's Multiple Range Test (DMRT) was used to differentiate between mean differences.

Statement of permissions and/or licenses for collection of plant or seed specimens
The authors declare that the cassava root specimens used in this study are publicly accessible cassava cultivar and we were given explicit written permission to use them for this research.

Plant guidelines
Experimental research and field studies on plants, including the collection of plant material comply with relevant institutional, national and international guidelines and legislation-Formal ethical approval is not required.

Bio-synthesis of silver nanoparticles
In this study, two different strains of Trichoderma species, T. harzianum and T. virens, were screened for the synthesis of AgNPs.Results showed that the biosynthesis of AgNPs was accomplished by combining 100 mL of 5 mM AgNO 3 solution with 5 g of Trichoderma species biomass or 100 mL of its supernatant.The implementation of this methodology produced the synthesis of AgNPs.The synthetization of AgNPs was confirmed by a color change from transparent to yellow and finally to reddish (Figs. 1 and 2) as was similarly observed when synthetizing AgNPs from T. virens.

Dynamic light scattering
The results obtained from the DLS showed a mean size of 64.92 and 41.78 nm, and a zeta potential of − 38.9 and + 39.14 mV for the AgNPs synthesized using T. virens biomass and supernatant, respectively.Whereas, 59.2 and 39.79 nm were recorded from AgNPs samples synthesized using T. harzianum biomass and supernatant, respectively; and mean zeta potentials of − 23.7 and − 36.5 mV, respectively (Table 1).The synthesized AgNPs with the smallest recorded average particle size was used for further analysis.

Scanning electron microscope analysis
The morphology of the AgNPs synthesized using T. harzianum supernatant was further analyzed using SEM.
The SEM image clearly showed the synthesis of particles which measured 60-75 nm (Fig. 3), confirming the development of silver nanostructures.

FE-TEM/STEM-EDS analysis
A comprehensive structural investigation of AgNPs was conducted using high-resolution transmission electron microscopy (HR-TEM).The HR image of the AgNP crystal structure displayed nanoparticles that were uniformly spherical in shape and had an estimated size of less than 50 nm (Fig. 4).EDS was performed to confirm the elemental distribution in AgNPs.The HAADF image and EDS mapping of AgNPs revealed a strong signal for   www.nature.com/scientificreports/Ag and was mainly localized in the core domain of AgNPs (red) (Fig. 5a,b).The EDS spectrum of nanoparticles showed major peak at 2.9846 keV for silver metal.The other metal ions, including carbon (C) and copper (Cu), also appeared in the EDS spectrum, which can detect X-rays generated from the substrate material of the TEM grid.

Determination of the antimicrobial activity and minimum inhibitory concentration
The antimicrobial activity of biosynthesized AgNPs against L. theobromae and F. solani was compared to the positive control (silver nitrate, carbendazim) and the negative control (water).The results indicated that the size of the fungi colonies decreased when increasing the concentration of AgNPs (Figs. 6, 7) until completely inhibiting the growth of L. theobromae at ≥ 58 µg mL −1 and F. solani at AgNPs ≥ 50 µg mL −1 .Meanwhile, AgNO 3 at an equal or larger dose than 120 µg mL −1 completely inhibited the growth of L. theobromae and F. solani (Table 2).

In vitro culture of Lasiodiplodia theobromae and Fusarium solani with AgNPs
To further test whether the development of L. theobromae or F. solani aerial hyphae growth was affected by AgNPs, additional experimentation was conducted under microscopy.The application of AgNPs caused a strong decrease in the percentage of healthy aerial hyphae of L. theobromae growth on the PDA plates, with only 67.50% by using 40 µg mL −1 , and reduced to 23.00% by using 50 µg mL −1 .Whereas the treatment of AgNO 3 showed a decrease of healthy aerial hyphae to 80.00% and to 56.00% when using the previous respective concentrations.
In the same trend, the percentage of healthy aerial hyphae of F. solani treated with AgNPs at 40 µg mL −1 and at 50 µg mL −1 decreased to 29.50 and 21.50% which resulted to be much more effective than using AgNO 3 (74.00and 42.00%).Moreover, the applied carbendazim at the same concentrations of AgNPs did not effectively inhibit hyphae growth as presented in Fig. 8.

Biochemical changes of mycelium using SR-FTIR spectroscopy
The SR-FTIR spectral data was analyzed to investigate biochemical changes in L. theobromae and F. solani mycelium after the application of the treatments with AgNPs and AgNO 3 .The results showed a clear separation of the chemical composition of mycelium between groups of the treated samples with 40 and 50 µg mL −1 of AgNPs in comparison to 50 µg mL −1 of AgNO 3 and to untreated.These results are supported by the principal component analysis (PCA) score plot (Fig. 9a) which is explained by PC1 (38%) and PC2 (9%) that are linked to loading.The positive loading PC1 at 1077 and 1027 cm −1 was the one that significantly characterized the untreated samples which present higher intensity in the polysaccharide region.The highest negative loading from the PC1 at 2962, 2927, 2854, 1658, 1642 and 1548 cm −1 corresponded with the negative score plot obtained from the AgNPs treated samples which presented significant intensities in the protein and lipids region (Fig. 9b).AgNPs treated samples showed differences in the spectra corresponding to the individual functional groups in three distinguishable regions (Fig. 10).The first region (3000-2800 cm −1 ) associated with mycelium composition which can be assigned to CH 2 and CH 3 stretch is mainly composed of lipids and fatty acids where the highest peaks were found at 2962, 2927, and 2854 cm −1 in the AgNPs treated samples.The second region (1700-1500 cm −1 ), composed by proteins and peptides with amide I and amide II, shows the highest peaks at 1658, 1642, and 1548 cm −1 in the AgNPs treated samples.The third region (1300-900 cm −1 ), which involves nucleic acid, phospholipids, polysaccharides, and carbohydrates, shows the highest peaks at 1155, 1079, and 1027 cm −1 from the untreated samples.

Effect of bio-synthetized AgNPs to control cassava root rot disease
The potential effects of the biosynthesized AgNPs on reducing disease severity on cassava root cultivar CMR 89 were studied.The disease severity caused by L. theobromae in samples treated with 50 µg mL −1 of AgNPs (1.75 ± 0.50) was significantly lower than the severity observed from samples treated with 100 µg mL −1 of AgNO 3 (2.50 ± 0.57) and from the control samples (5.00 ± 0.00).The AgNPs treatment had no significant different results compared to the 4 mg mL −1 carbendazim treatment (1.50 ± 0.57).Moreover, the AgNPs treatment also showed the lowest disease severity caused by F. solani as 1.00 ± 0.00 followed by carbendazim (1.25 ± 0.50) and AgNO 3 (1.50 ± 0.57) which were all significantly effective compared to control (Table 3, Figs.11 and 12).

Discussion
Bio-reduction of AgNO 3 into AgNPs after the addition of T. harzianum supernatant was confirmed by its change in color.Initially, after the addition of the T. harzianum supernatant, the color turned to pale yellow and continued to change to light brown as the incubation time was increasing.After 24 h of incubation, the solution changes to deep brown color.A complete color change was seen at 48 h after incubation when no further color change was observed which indicated that all the AgNO 3 was completely reduced to AgNPs.This change in color is an indication of the AgNP formation (reduction process) due to the excitation of the surface plasmon resonance (SPR) of silver nitrate 34 .Proteins, essential amino acids, and other substances present in T. harzianum supernatant serve as natural reducing and capping agents in the synthesis of AgNPs for reducing silver nitrate to AgNPs 35 .T. harzianum are filamentous fungi known for their ability to produce various enzymes, including reducing enzymes, that can catalyze the reduction of metal ions, such as Ag + ions from silver nitrate, into their metallic  www.nature.com/scientificreports/nanoparticle form.The reducing enzymes present in the supernatant, such as nitrate reductase, act as catalysts to facilitate the reduction of Ag + ions (silver cations) to metallic silver (Ag 0 ).The enzymes provide electrons to the Ag + ions, converting them into AgNPs.The DLS analysis measured the particle size and diffusion coefficient of the nanoparticles, confirming the synthesis of AgNPs using T. harzianum supernatant.The size of AgNPs using T. harzianum supernatant was smaller than using the other proposed treatments (Table 1) which may be caused by the capping effect of nanoparticles from the T. harzianum metabolites.The size of a nanoparticle influences its physiological reaction, distribution, and elimination.According to the DLS, the dispersion only contains nanoparticles with an average diameter size of 39.79 nm.The minimum particle size that can be measured by the DLS is smaller than the SEM which is convenient in this study (Fig. 3).This can be explained as the DLS considers the capping and the lowering agents when determining the size.It is important to note that, in addition to size, nanoparticle stability is critical for promoting biological activity 36 .The zeta potential quantifies the electrical charge on the surface of nanoparticles.Since higher zeta potential nanoparticles reject one another, they are less likely to form nano agglomerations and are more stable 37 .Particles with zeta potential greater than + 30 mV or less than − 30 mV are assumed to be more stable than those with intermediate zeta potentials 38 .The bio-synthesized AgNPs had a zeta potential of + 39.14 mV, indicating that they were highly stable.The electrostatic attraction of positively charged capping agents in nanoparticles caused the system to generate a positive zeta potential.The biological activity results showed that the AgNPs presented potential for the control of L. theobromae and F. solani since mycelium growth was reduced.The development of L. theobromae or F. solani aerial hyphae growth were affected by AgNPs-based treatment.Treatments using AgNO 3 , and carbendazim also resulted in the inhibition of L. theobromae and F. solani at higher concentrations as shown in Table 2; whereas, the AgNPs at lower concentrations can effectively reduce mycelium growth (Figs. 4 and 5).AgNPs are frequently used against crop diseases because of their antagonistic activity against a broad range of phytopathogens 22 .In this study, the bio-synthesized AgNPs showed inhibitory activity against hypha growth, remarking their potential application against root rot disease.For further understanding, an analysis on the biochemical changes of the pathogens mycelium using SR-FTIR microspectroscopy was performed.The lipid region was found at 2962, 2927, and 2854 cm −1 in the spectral data obtained from the AgNPs treated samples which principally provided asymmetric and symmetric stretching of CH 3 and CH 2. Lipids are the composition of fungus mycelium used to formulate edible coatings.Lipids are also the most abundant respiratory substrates in extra-radical mycelium, and they play critical roles in the fungus carbon metabolism and its transport 39 .Furthermore, lipid transporters in fungal membrane associated to biosynthetic and metabolic pathways contribute to virulence mechanisms that are important targets for protecting cell from environmental stress as well as for protein resistance, biofilm formation, and extracellular polysaccharide 40,41 .According to Walley et al. 42 , fatty acids also modulate a variety of signal transduction pathways triggered by environmental and developmental stimuli 42 .Our results are in accordance to the previous analysis conducted by Shapaval et al. 43 .The authors indicated that the fatty acid profile of the fungi Mucor plumbeus on different carbon source mediums led to changes in the FTIR spectra (3000-2800 cm −1 ) that revealed contents of unsaturated fatty acids.Similarly, Walley et al. 42 reported that the increase in free fatty acid levels depends on stress responses which play a crucial role in pathogen membranes composition, and act as protected signaling molecules that is transported across eukaryotic cells for immune responses against biotic or abiotic stress.In addition, the membrane damage integrity mechanism contributes to deliver an increase of levels in the acyl chain, causing the peaks in the 3000-2800 cm −1 region to increase as well.The absorption band located at 1658, 1641, and 1548 cm −1 are related to the protein peptides; Amide I (1600-1690 C=O stretching vibration) and Amide II (1480-1575; CN and NH bending vibration) were assigned to alpha helical structures, lysine structure, and N-terminal amino group 44,45 .The group of amino acids, proteins, and polypeptide found in fungi are important for the well-functioning of their immune system, their adaptation to ecological niches or to severe environments, and for producing fungal immunomodulatory proteins (FIPs) and small secreted proteins (SSPs) 46,47 .In addition, fungi secrete diverse groups of amino acid groups through small proteins to be essential for their virulence 48 .Our results are in accordance with Skoneczny and Skoneczna 49 .
The authors reported that eukaryotes, including yeast and fungi, can adjust their transcriptional response, allowing adaptation to various chemical and physical stresses by developing pathways of mitogen-activated protein kinase activation.Similarly, Künzler 50 reported that the most important defense mechanism of fungi is a chemical enzyme based on secondary metabolites, peptides, and proteins which are usually stored and produced within the fungal cells.The third region, which references polysaccharides and carbohydrates of fungi mycelium, showed higher peaks at 1155, 1079, and 1027 cm −1 from spectral readings of the untreated samples.These results determined that the polysaccharides and carbohydrates as cell wall C-O-C shifted to lower lectures in the AgNPs  www.nature.com/scientificreports/treated samples.This observation demonstrates that the AgNPs causes damage to mycelium since there is a detected peak of spectra representing fungi cell membrane 51,52 .These suggestions agree with the report of Mihoubi et al. 51 .who observed DNA damages of yeast demonstrated by the intensity drop of peaks at 1048 cm −1 and 1079 cm −1 indicating a loss of nucleic acids 52 .Biochemical changes in mycelium can also be associated with the expression or production of pathogen virulence factors 52 .The virulence factor changes can affect the pathogen ability to infect, colonize, or evade the host immune responses 53 .Such changes lead to less virulent or less

Figure 1 .
Figure 1.Color changes during the biosynthesis of AgNPs from 5 mM of silver nitrate solution with 5 g of T. harzianum biomass at different reaction times: (a) silver nitrate solution without T. harzianum biomass, (b) biosynthesis of AgNPs at 0 h, (c) biosynthesis of AgNPs at 24 h, and (d) biosynthesis of AgNPs at 48 h.

Figure 2 .
Figure 2. Color changes during the biosynthesis of AgNPs from 5 mM of silver nitrate solution with T. harzianum supernatant at different reaction times: (a) silver nitrate solution without T. harzianum supernatant, (b) biosynthesis of AgNPs at 0 h, (c) biosynthesis of AgNPs at 24 h, and (d) biosynthesis of AgNPs at 48 h.

Figure 3 .
Figure 3. Scanning electron microscope (SEM) image of silver nanoparticles synthesized by Trichoderma harzianum supernatant from silver nitrate.

Figure 4 .
Figure 4. High resolution transmission electron microscopy (HR-TEM) image of silver nanoparticles synthesis by Trichoderma harzianum supernatant.

Figure 8 .
Figure 8.Quantification of healthy aerial hyphae on media containing AgNPs, AgNO 3 , and Carbendazim.(a) The healthy aerial hyphae rate of Lasiodiplodia theobromae and (b) the healthy aerial hyphae rate of Fusarium solani.Means in the graph followed by different letters is a are significantly different, ns = not statistically significant according to DMRT at P = 0.05.

Table 1 .
The size (nm) and zeta potential (mV) of biosynthesis AgNPs.Data are shown as ± mean standard deviation of four replications.

Table 2 .
Inhibition rate and minimum inhibitory concentration (MIC) at different concentrations of the AgNPs against Lasiodiplodia theobromae and Fusarium solani causing cassava root rot disease.The meaning of the different letters (a, b, c) indicated significant difference via DMRT at P = 0.05.

Table 3 .
Effectiveness of biosynthesized silver nanoparticles on the disease severity of cassava root rot caused by Lasiodiplodia theobromae and Fusarium solani.The meaning of the different letters (a,b,c) indicated significant difference via DMRT at P = 0.05.